The aquatic ecosystem, a good environment for the horizontal transfer of antimicrobial resistance and virulence-associated factors among extended spectrum beta-lactamases producing E. coli

One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum β-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 × 10−1 ± 5 × 10−1 , being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 × 10−4 ± 2.85 × 10−4 and 9.61 × 10−4 ± 1.96 × 10−3 , respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs

The aquatic ecosystem, a good environment for the horizontal transfer of antimicrobial resistance and virulence-associated factors among extended spectrum ß-lactamases producing E. coli

One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum β-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 × 10−1 ± 5 × 10−1 , being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 × 10−4 ± 2.85 × 10−4 and 9.61 × 10−4 ± 1.96 × 10−3 , respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs

Multidrug-Resistant Bacteria Isolated from Different Aquatic Environments in the North of Spain and South of France

Due to the global progress of antimicrobial resistance, the World Health Organization (WHO) published the list of the antibiotic-resistant “priority pathogens” in order to promote research and development of new antibiotics to the families of bacteria that cause severe and often deadly infections. In the framework of the One Health approach, the surveillance of these pathogens in different environments should be implemented in order to analyze their spread and the potential risk of transmission of antibiotic resistances by food and water. Therefore, the objective of this work was to determine the presence of high and critical priority pathogens included in the aforementioned list in different aquatic environments in the POCTEFA area (North Spain–South France). In addition to these pathogens, detection of colistin-resistant Enterobacteriaceae was included due its relevance as being the antibiotic of choice to treat infections caused by multidrug resistant bacteria (MDR). From the total of 80 analyzed samples, 100% of the wastewater treatment plants (WWTPs) and collectors (from hospitals and slaughterhouses) and 96.4% of the rivers, carried antibiotic resistant bacteria (ARB) against the tested antibiotics. Fifty-five (17.7%) of the isolates were identified as target microorganisms (high and critical priority pathogens of WHO list) and 58.2% (n = 32) of them came from WWTPs and collectors. Phenotypic and genotypic characterization showed that 96.4% were MDR and resistance to penicillins/cephalosporins was the most widespread. The presence of bla genes, KPC-type carbapenemases, mcr-1 and vanB genes has been confirmed. In summary, the presence of clinically relevant MDR bacteria in the studied aquatic environments demonstrates the need to improve surveillance and treatments of wastewaters from slaughterhouses, hospitals and WWTPs, in order to minimize the dispersion of resistance through the effluents of these areas

Prevalence of integrons and insertion sequences in ESBL-producing E-coli isolated from different sources in Navarra, Spain

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26–ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1–IS26–IS903–ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and β-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments

The CO2-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases

Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5¿10% CO2 necessity for growth (CO2-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1¿4 strains. Although molecular tests are replacing phenotypic methods, CO2-dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO2-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO2 to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO2-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO2-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO2-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO2-independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media. Finally, we also found that insertion of a heterologous active CAII into B. ovis reverted the CO2-dependence but did not alter its virulence in the mouse model. These results allow a better understanding of central aspects of Brucella metabolism and, in the case of B. ovis, provide tools for large-scale production of diagnostic antigens and vaccines

The aquatic ecosystem, a good environment for the horizontal transfer of antimicrobial resistance and virulence-associated factors among extended spectrum ß-lactamases producing E. coli

One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum β-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 × 10−1 ± 5 × 10−1 , being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 × 10−4 ± 2.85 × 10−4 and 9.61 × 10−4 ± 1.96 × 10−3 , respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs

The aquatic ecosystem, a good environment for the horizontal transfer of antimicrobial resistance and virulence-associated factors among extended spectrum beta-lactamases producing E. coli

One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum β-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 × 10−1 ± 5 × 10−1 , being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 × 10−4 ± 2.85 × 10−4 and 9.61 × 10−4 ± 1.96 × 10−3 , respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs

Vigilancia, prevención, diagnóstico y tratamiento de las resistencias a antibióticos B-lactámicos desde la perspectiva One Health

Before the outbreak of the pandemic Covid-19, one of the greatest threats to medicine in the 21st century was and continues to be the antimicrobial resistances (AMR). In order to address this problem with a global perspective and taking into account the strategic lines proposed by the Spanish National Antibiotic Resistance Plan (PRAN), the general objective of this work has been to approach the surveillance, prevention, diagnosis and treatment of resistance to antibiotics, with special emphasis on the extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL). The first objective focused on the surveillance of the resistances in different environments. In Chapter 1 it was reported the isolation and characterization of priority pathogens according to the WHO list from rivers, wastewater treatment plants (WWTPs) and collectors in northern Spain and southern France (POCTEFA area). The results showed that 100% of the WWTPs and collectors and 96.4% of the rivers carried resistant bacteria against at least one of the following antibiotic families: β-lactams, carbapenems, vancomycin and colistin. More than a half of the 55 isolated strains come from wastewater environments and multidrug resistances (MDR) were observed in 96.4% of them, with penicillin/cephalosporin resistance being the most widespread. In agreement with that, in Chapter 2 we performed the phylogenetic characterization of ESBL-producing E. coli strains, isolated from animal, environmental and human environment in the present and previous studies. Important clonal complexes (23CC, 10CC, 131CC and 38CC) related to the spread of β-lactam antibiotic resistance genes were found, but none of them was present in all sources. However, wastewater environments housed these 4 CCs and showed identical profiles to strains isolated from other environments, showing the importance that water acquire in the dissemination of these resistances. Abstract 24 The second objective addressed the study of the diffusion of resistance among the different ecosystems. Therefore, in Chapter 3 the characterization of integrons and insertion sequences was carried out. Results showed the wide presence and dissemination of intI1 (92%), IS26 (99.4%) as well as the genetic pattern IS26-ISEcp1 (related with the pathogenic clone 131CC) that was present in 22% of the strains. The coexistence of various types of integrons and insertion sequences suggests a possible risk of spread of resistance between different environments. Meanwhile, in Chapter 4 it was analysed the genetic profiles associated with virulence factors, as well as the conjugative capacity presented by these E. coli strains. Virulence genes fimA, papC, and aer were detected in all environments, papG III was mainly associated with clinical strains, and wastewater was a point of diffusion for cnf1 and hlyA genes. In addition, isolated strains from aquatic environments showed a significantly higher conjugation frequency than those coming from farms and food, pointing one more time the importance that the aquatic environment acquires for the exchange of genes and resistance to antibiotics. The third objective consisted of evaluating a proposal for the prevention of AMR in animal environment. Thus, in Chapter 5 it was assessed the antimicrobial activity of silver nanomaterials to be added in feed as an alternative to the use of antibiotics in animal production. The results showed that C3 product has antimicrobial activity against a wide spectrum of bacteria (including sensitive and resistant ones), with a higher bactericidal activity against Gram negative bacteria (lower concentration needed and faster reduction of viable microorganisms than in the case of S. aureus). Finally, in the fourth objective, diagnostic and treatment alternatives for infections caused by ESBL-producing Enterobacteriaceae were addressed. In Chapter 6 a cefotaxime hydrolysis protocol has been defined that allows rapid detection of ESBL- Abstract 25 producing strains (CTX-M1 type) by using the VITEK®-MS RUO (bioMérieux). Secondly, in Chapter 7 two treatment alternatives have been tested; on the one hand, the activity of two antimicrobial peptides derived from lactoferricin (P4-1 and P2-15) was evaluated against ESBL-producing E. coli. Despite subinhibitory concentrations of both peptides in the presence of 1 µg/ml of clavulanic acid managed to reduce the MIC of the antibiotic, none of them achieved sensitization to amoxicillin. On the other hand, the antibacterial activity of the metabolites produced by 3 lactic acid bacteria (C1, A1 and C34) of animal origin was studied, confirming that they were capable of inhibiting the growth of ESBL-producing E. coli, being L. plantarum C1 the most active one, showing a genetic cluster compatible with the plantaricin-type bacteriocin. The data obtained in this Doctoral Thesis have shown that it is necessary to carry out a more effective control of the presence of MDR bacteria in the aquatic environment, implementing surveillance of relevant clonal complexes that act as markers for the presence of resistant bacteria as well as more effective wastewater treatment programs to stop the dispersion. In relation to the animal environment, a silver nanomaterial was positively evaluated as possible feed additive to replace or reduce the use of antibiotics in animal production. Finally, with regard to human health, a rapid diagnostic protocol for the detection of blaCTX-M1-producing Enterobacteriaceae was developed using the VITEK®-MS RUO equipment. In addition, the antimicrobial activity of the three tested LAB was confirmed, but further studies are necessary before considering their possible therapeutic application

Vigilancia, prevención, diagnóstico y tratamiento de las resistencias a antibióticos B-lactámicos desde la perspectiva One Health

Before the outbreak of the pandemic Covid-19, one of the greatest threats to medicine in the 21st century was and continues to be the antimicrobial resistances (AMR). In order to address this problem with a global perspective and taking into account the strategic lines proposed by the Spanish National Antibiotic Resistance Plan (PRAN), the general objective of this work has been to approach the surveillance, prevention, diagnosis and treatment of resistance to antibiotics, with special emphasis on the extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL). The first objective focused on the surveillance of the resistances in different environments. In Chapter 1 it was reported the isolation and characterization of priority pathogens according to the WHO list from rivers, wastewater treatment plants (WWTPs) and collectors in northern Spain and southern France (POCTEFA area). The results showed that 100% of the WWTPs and collectors and 96.4% of the rivers carried resistant bacteria against at least one of the following antibiotic families: β-lactams, carbapenems, vancomycin and colistin. More than a half of the 55 isolated strains come from wastewater environments and multidrug resistances (MDR) were observed in 96.4% of them, with penicillin/cephalosporin resistance being the most widespread. In agreement with that, in Chapter 2 we performed the phylogenetic characterization of ESBL-producing E. coli strains, isolated from animal, environmental and human environment in the present and previous studies. Important clonal complexes (23CC, 10CC, 131CC and 38CC) related to the spread of β-lactam antibiotic resistance genes were found, but none of them was present in all sources. However, wastewater environments housed these 4 CCs and showed identical profiles to strains isolated from other environments, showing the importance that water acquire in the dissemination of these resistances. Abstract 24 The second objective addressed the study of the diffusion of resistance among the different ecosystems. Therefore, in Chapter 3 the characterization of integrons and insertion sequences was carried out. Results showed the wide presence and dissemination of intI1 (92%), IS26 (99.4%) as well as the genetic pattern IS26-ISEcp1 (related with the pathogenic clone 131CC) that was present in 22% of the strains. The coexistence of various types of integrons and insertion sequences suggests a possible risk of spread of resistance between different environments. Meanwhile, in Chapter 4 it was analysed the genetic profiles associated with virulence factors, as well as the conjugative capacity presented by these E. coli strains. Virulence genes fimA, papC, and aer were detected in all environments, papG III was mainly associated with clinical strains, and wastewater was a point of diffusion for cnf1 and hlyA genes. In addition, isolated strains from aquatic environments showed a significantly higher conjugation frequency than those coming from farms and food, pointing one more time the importance that the aquatic environment acquires for the exchange of genes and resistance to antibiotics. The third objective consisted of evaluating a proposal for the prevention of AMR in animal environment. Thus, in Chapter 5 it was assessed the antimicrobial activity of silver nanomaterials to be added in feed as an alternative to the use of antibiotics in animal production. The results showed that C3 product has antimicrobial activity against a wide spectrum of bacteria (including sensitive and resistant ones), with a higher bactericidal activity against Gram negative bacteria (lower concentration needed and faster reduction of viable microorganisms than in the case of S. aureus). Finally, in the fourth objective, diagnostic and treatment alternatives for infections caused by ESBL-producing Enterobacteriaceae were addressed. In Chapter 6 a cefotaxime hydrolysis protocol has been defined that allows rapid detection of ESBL- Abstract 25 producing strains (CTX-M1 type) by using the VITEK®-MS RUO (bioMérieux). Secondly, in Chapter 7 two treatment alternatives have been tested; on the one hand, the activity of two antimicrobial peptides derived from lactoferricin (P4-1 and P2-15) was evaluated against ESBL-producing E. coli. Despite subinhibitory concentrations of both peptides in the presence of 1 µg/ml of clavulanic acid managed to reduce the MIC of the antibiotic, none of them achieved sensitization to amoxicillin. On the other hand, the antibacterial activity of the metabolites produced by 3 lactic acid bacteria (C1, A1 and C34) of animal origin was studied, confirming that they were capable of inhibiting the growth of ESBL-producing E. coli, being L. plantarum C1 the most active one, showing a genetic cluster compatible with the plantaricin-type bacteriocin. The data obtained in this Doctoral Thesis have shown that it is necessary to carry out a more effective control of the presence of MDR bacteria in the aquatic environment, implementing surveillance of relevant clonal complexes that act as markers for the presence of resistant bacteria as well as more effective wastewater treatment programs to stop the dispersion. In relation to the animal environment, a silver nanomaterial was positively evaluated as possible feed additive to replace or reduce the use of antibiotics in animal production. Finally, with regard to human health, a rapid diagnostic protocol for the detection of blaCTX-M1-producing Enterobacteriaceae was developed using the VITEK®-MS RUO equipment. In addition, the antimicrobial activity of the three tested LAB was confirmed, but further studies are necessary before considering their possible therapeutic application

Multidrug-Resistant Bacteria Isolated from Different Aquatic Environments in the North of Spain and South of France

Due to the global progress of antimicrobial resistance, the World Health Organization (WHO) published the list of the antibiotic-resistant “priority pathogens” in order to promote research and development of new antibiotics to the families of bacteria that cause severe and often deadly infections. In the framework of the One Health approach, the surveillance of these pathogens in different environments should be implemented in order to analyze their spread and the potential risk of transmission of antibiotic resistances by food and water. Therefore, the objective of this work was to determine the presence of high and critical priority pathogens included in the aforementioned list in different aquatic environments in the POCTEFA area (North Spain–South France). In addition to these pathogens, detection of colistin-resistant Enterobacteriaceae was included due its relevance as being the antibiotic of choice to treat infections caused by multidrug resistant bacteria (MDR). From the total of 80 analyzed samples, 100% of the wastewater treatment plants (WWTPs) and collectors (from hospitals and slaughterhouses) and 96.4% of the rivers, carried antibiotic resistant bacteria (ARB) against the tested antibiotics. Fifty-five (17.7%) of the isolates were identified as target microorganisms (high and critical priority pathogens of WHO list) and 58.2% (n = 32) of them came from WWTPs and collectors. Phenotypic and genotypic characterization showed that 96.4% were MDR and resistance to penicillins/cephalosporins was the most widespread. The presence of bla genes, KPC-type carbapenemases, mcr-1 and vanB genes has been confirmed. In summary, the presence of clinically relevant MDR bacteria in the studied aquatic environments demonstrates the need to improve surveillance and treatments of wastewaters from slaughterhouses, hospitals and WWTPs, in order to minimize the dispersion of resistance through the effluents of these areas